A new micropolydesmoid millipede of the genus Eutrichodesmus Silvestri, 1910 from Cambodia, with a key to species in mainland Southeast Asia (Diplopoda, Polydesmida, Haplodesmidae)

Abstract The micropolydesmoid millipede family Haplodesmidae is here recorded from Cambodia for the first time through the discovery of the first, new species of the genus Eutrichodesmus Silvestri, 1910: E. cambodiensissp. nov. This new species is described from two limestone habitats in Kampot Province, based on abundant material. It is easily distinguished from all related congeners by the following combination of characters: body greyish-brown; limbus roundly lobulate; solenomere partially divided from acropodite by a digitiform lobe, but without hairpad. Brief remarks on the previously-proposed “pecularis-group” are provided and a second group, the “demangei-group”, is established and discussed on the basis of morphological evidence, updating the number of recognised species groups of Eutrichodesmus to two. Detailed morphological illustrations, photographs and a distribution map, as well as remarks on its habitat and mating behaviour of the new species are presented. Furthermore, the current distributions of all 55 presently-known species of Eutrichodesmus are provided and a key to all 23 species that occur in mainland Southeast Asia is given.


Introduction
Previous and recent studies on millipedes in the Kingdom of Cambodia have revealed at least 23 species from 17 genera, 12 families and eight orders (Likhitrakarn et al. 2015(Likhitrakarn et al. , 2020Golovatch 2018). Although the Polydesmida Pocock, 1887 is the most diverse order of Diplopoda worldwide, only two polydesmidan families have hitherto been reported from Cambodia: Cryptodesmidae and Paradoxosomatidae. The Cryptodesmidae is typically considered as "micropolydesmoid" due to small body sizes of its species. The group is represented in Cambodia by only two species: Trichopeltis kometis (Attems, 1938) and Circulocryptus kompantsevi Golovatch, 2018. The former species was originally described from Kratié Province (Attems 1938), but it has since been recorded from Vietnam and Laos as well (Golovatch and Akkari 2016). Circulocryptus kompantsevi has recently been described from a rain-and-cloud forest at about 1,000 m above sea-level (AMSL) in the Phnom Bokor National Park, Kampot Province (Golovatch 2018). Surprisingly, no other micropolydesmoid families (e.g. Haplodesmidae or Pyrgodesmidae), both quite diverse and common in Indochina, have been recorded from Cambodia yet. Not only the micropolydesmoids, but also the genera Desmoxytes Chamberlin, 1923, Antheromorpha Jeekel, 1968and Tylopus Jeekel, 1968, all in the family Paradoxosomatidae and all quite diverse and common in the neighbouring countries, also appear to be poorly represented in Cambodia. This strongly contrasts with the adjacent parts of Indochina where more than 20 micropolydesmoid species have been discovered in Laos, Thailand and Vietnam over the last few years Liu et al. 2017;Golovatch 2018;Likhitrakarn et al. 2019). Amongst these micropolydesmoids, many are quite rare and most are known only from their original descriptions.
The micropolydesmoid genus Eutrichodesmus Silvestri, 1910 is amongst the most speciose not only in Haplodesmidae, but also in the entire order Polydesmida. Its distribution ranges from southern Japan in the north, through Taiwan, continental China and mainland Southeast Asia, to Indonesia and Vanuatu in the south (Liu and Wynne 2019;Golovatch and Liu 2020). Eutrichodesmus currently comprises 54 recognised species (Sierwald and Spelda 2019), of which over half are known from continental China alone, whereas 22 species are restricted to mainland Southeast Asia. Golovatch et al. (2009aGolovatch et al. ( , 2009b provided the most thorough and basic treatment of the group. However, since then, the number of known species has increased almost three-fold (Golovatch et al. 2009bMakhan 2010;Liu et al. 2013Liu et al. , 2017Liu and Wynne 2019). This invites an update and a modern key.
We have recently conducted surveys in southern Cambodia with emphasis on the biodiversity of limestone karsts. A considerable amount of material has been collected and become available for study. As a result, several new species from different millipede groups have been revealed and mostly already described: Plusioglyphiulus Silvestri, 1923 andTrachyjulus Peters, 1864 (Cambalopsidae, Spirostreptida), as well as Tylopus and Orthomorpha Bollman, 1893 (Paradoxosomatidae, Polydesmida). The present paper is devoted to the description of a new Eutrichodesmus, the first Haplodesmidae to be recorded from Cambodia. We also provide an identification key to and update the distributions of all known species of Eutrichodesmus.

Material and methods
The material for this contribution was collected during surveys on freshwater and terrestrial invertebrates in Cambodia, conducted jointly by researchers from the Inland Fisheries Research & Development Institute of Cambodia (IFReDI) and several Thai specialists. Since the expeditions started (from 2018 until now), large collections of millipedes have become available, also representing the first reference collections in Cambodia.

Specimen collecting and preservation
All specimens were hand-collected from limestone habitats in Cambodia. Live animals were photographed using a Nikon D700, equipped with an AF-S VR Micro-Nikkor 105 mm lens in the field. Some mating pairs were observed at the type locality and some were brought back to the laboratory for further behavioural observations. Specimens were euthanised, based on AVMA guidelines for the euthanasia of animals (American Veterinary Medical Association 2020) and then mostly stored in 70% (v/v) ethanol for morphological study and, partly, in 95% (v/v) ethanol for molecular analysis. Latitude, longitude and elevation were obtained from a Garmin GPSMAP 60 CSx and all coordinates and elevations were double-checked with Google Earth to confirm the precise location.

Morphological descriptions
All specimens of the new species were carefully examined for non-gonopodal and gonopodal characteristics using stereo and compound light microscopes. For some male specimens, the gonopods were carefully dissected and then mounted on a slide with DPX/ balsam. The morphological terminology used in this study follows that of previous publications (Golovatch et al. 2009a(Golovatch et al. , 2009bHoffman 1977aHoffman , 1977bLiu and Tian 2013;Liu et al. 2017). Details of gonopodal terminology are shown in the section "Abbreviations used in descriptions" below.
The holotype and some paratypes are deposited in the Chulalongkorn University Museum of Zoology (CUMZ-hpd0001 and CUMZ-hpd0002). Some paratypes are housed in the collections of the Inland Fisheries Research and Development Institute (CIFI), Cambodia and the Zoological Reference Collection (ZRC) of the Lee Kong Chian Natural History Museum, Singapore.
All available literature sources, especially the original descriptions, were critically accessed in order to compare morphological characters to all known species. Positional and directional terms for gonopod descriptions follow Srisonchai et al. (2018).

Illustrations
Drawings were sketched under a stereomicroscope and a light microscope. All plates of figures were generated and edited using Adobe Photoshop CS6 to adjust the colour and brightness. The distribution map was modified from Willett et al. (2015).

Abbreviations used in descriptions
As the previous studies of gonopods from different authors are quite variable, ranging from a brief description to several deeply detailed ones, we chose to follow the comprehensive gonopod terminology from Golovatch et al. (2009bGolovatch et al. ( , 2015, Hoffman (1977aHoffman ( , 1977b and Liu et al. (2017).
Abbreviations: cn = cannula, cx = coxa, dp = distofemoral process, sg = seminal groove; acropodite = the apical part of gonopod that starts from a prominent cingulum (the end of the femorite); solenomere = an independent part of the gonopod acropodite that carries the seminal groove, with or without hairpad, completely or partly fused to the acropodite; telopodite = the main part of the gonopod pivoting on the coxa, including the prefemur, femorite and acropodite.  Silvestri, 1910 All species included. The genus Eutrichodesmus currently contains 55 species, including the new one described herein, see Table 1.
Recorded distributions of all known species. Based on all the recent literature and excluding the newly-described species, the genus Eutrichodesmus is widely distributed in southern Japan, Taiwan, southern China, mainland Southeast Asia (Malaysia, Laos, Thailand and Vietnam), Indonesia (Sulawesi) and Melanesia (Vanuatu) (Golovatch et al. 2009a(Golovatch et al. , 2009bLiu et al. 2017;Golovatch and Liu 2020; see Table 1). No Eutrichodesmus species have hitherto been reported from Cambodia.

Species
Diagnosis. Body with incomplete volvation; metaterga with three transverse rows of regular and round tubercles, but no mid-dorsal projection (outgrowth) on metaterga; distofemoral process on gonopod telopodite very short, inconspicuous. Similar in all these characters to E. griseus Golovatch et al., 2009, but differs in having (1) live specimens and freshly preserved material pale greyish-brown or pale brown in colour; (2) the limbus crenulate, but not spinulate, crenulations being slightly longer than broad; (3) the acropodite curved and long, unciform, attenuated near tip; with a free solenomere starting from about midway; and (4) the solenomere digitiform, papillate, without hairpad.
Colour (Fig. 1). Live specimens pallid greyish-brown or brown: head grey; antennae pale brown; collum, metaterga and paraterga greyish-brown; surface below paraterga, prozonae, sterna and legs brown. Specimens in alcohol after six months of presevration nearly the same in colour as in life. Body ( Fig. 2A). General appearance as in Fig. 2A. Body long and slender. Adults with 20 rings. Volvation incomplete because of a slender body and short paraterga.
Head (Fig. 2B, C). Slightly transverse, wider than high, densely pilose, not covered with collum from above. Vertex microvillose and microgranulate. A pair of small, poorly separated, paramedian knobs above antennal sockets. Isthmus between antennae ca. 1.3 times as wide as diameter of antennal socket. Epicranial suture deep and conspicuous. Labrum and genae sparsely setose.
Paraterga (Figs 2A, E-F; 3A-C, E; 5W). Broad, slightly sloping down. Tip round and directed ventrolaterad. Paraterga 2 enlarged, in situ more strongly sloping down than on other rings, with four or five conspicuous lobules. Paraterga 3 and 4 shorter, narrower than others; each with four conspicuous lobules. Paraterga 5-18 mostly with four lobules, some rings with five ones.
Sterna (Fig. 3D). Narrow. Longitudinal depression between coxae in most body rings deep and narrow, only in ring 7 quite deep and wide for accommodating the shafts of gonopods. Transverse depression deep and wide.
Gonopod aperture. Very large, transversely ovoid, subequal to width of prozonite. Gonopods (Figs 4; 7T). Shafts when retracted reaching the anterior part of sternum 7 (base of legs 8). Coxa (cx) large and stout, subquadrate, microgranulate, with a few short setae distolaterally. Cannula (cn) simple, conspicuous, curved and slender, swollen at base, inserted into a small depression at base of telopodite on posteromedial side. Telopodite suberect; basal half (= prefemoral part) nearly straight; distal half curved. Distofemoral process (dp) very short, located at about midway of telopodite, triangular, dentate. Acropodite conspicuous, with neither a lobe nor a process, distally slightly attenuated and forming a hook-like tip, directed and curved mesad. Solenomere partially separated from acropodite, conspicuous, digitiform, papillate, originating at ca. 3/4 height of telopodite beyond distofemoral process; rather short, tip in situ directed anteriad, apically with a large papilla which is more conspicuous than other papillae. Seminal groove (sg) conspicuous, thick, running entirely on mesal surface of telopodite, terminating without hairpad by opening on the large papilla of solonomere.
Remarks. Although the genital characters of females have not been used for taxonomic purposes in the present study, all females were examined. In all cases, the female non-genital characters were found similar to those found in males. The only difference which can be clearly seen using both live and preserved material is that females are apparently broader and longer than males (Fig. 1C).
The general colouration does not show any variability and the paratypes do not differ significantly from the holotype. Across the type series of the new species, there was little intrapopulational variation in the number of tubercles on the collum and of lobes on the paraterga: an intermediate row (third or middle row) of the collum usually showed 1+1 tubercles, only sometimes 0+1 or 1+0 tubercles; paraterga of most specimens usually had four conspicuous lobes, only sometimes five. However, all these variations in most of the non-gonopodal characters were minor, neither significant nor consistent enough to be useful for taxonomic purposes, at least in the species under consideration. Little can be said about interpopulational variation in the new species because no variation has been noted between the two examined populations and no other specimens living at and around these two locations have been found.
Notably, E. cambodiensis sp. nov. shows a slightly elevated anterior margin of the collum (Figs 2A, 3A). As this can easily be seen also in E. griseus, it is consistent with what Golovatch et al. (2009b) found. Currently, only these two Eutrichodesmus species have the collum elevated in the anterior part, this strongly resembling the typical condition in the micropolydesmoid family Pyrgodesmidae.
The new species has the same characters as found in a bunch of congeners and shares the combination: adults with 20 body rings; body with incomplete volvation; metaterga without mid-dorsal projections, with three transverse rows of tubercles; and gonopod telopodite with a distofemoral process. All above characters are present in E. basalis Golovatch et al., 2009;E. curticornis Golovatch et al., 2009;E. demangei Silvestri, 1910;E. filisetiger Golovatch et al., 2009;E. gremialis (Hoffman, 1982); E. griseus, E. multilobatus Golovatch et al., 2009;E. nadan Golovatch et al., 2016;E. parvus Liu &Wesener, 2017 andE. regularis Golovatch et al., 2009 (see also Key and Table 2). Even though some traits have been observed, shared, especially in the gonopodal telopodite, between-species differences are always marked. With respect to the most relevant feature which lies in certain details of gonopodal structure, E. cambodiensis sp. nov. seems to be morphologically more similar to E. griseus than to any other congener, in particular in having a very short distofemoral process and the solenomere partly separated from the acropodite by forming a conspicuous lobe.
Distribution and habitat. It is worth noting that the new species was found only at the two sites. Surveys of other limestone and sandstone habitats surrounding the type locality (Kampong Trach) over a period of approximately two years have revealed no further specimens (Fig. 10). In showing a distribution of only two locations in a small and isolated limestone area, the new species can be suggested as being not only endemic to Cambodia, but also indigenous in Kampong Trach.
All specimens of the new species were hand-collected and found walking on humid rock walls of limestone caves (Fig. 9A). The vast majority of millipedes were seen crawling on humid rocks, whereas only a minor part was found slowly walking on vegetation, shaded holes and rock crevices during the daytime (Fig. 9B, C, E). It is important to note that specimens were commonly found under herb patches in a slightly shaded moist rock where the plant genus Epithema Blume, 1826 (family Gesneriaceae) created a mass of roots and thin litter layer on the soil in the hole (Fig. 9C). This is probably a particular microhabitat for E. cambodiensis sp. nov. Furthermore, we noted a co-occurrence between E. cambodiensis sp. nov. and the abundant Hypselostoma cambodjense Benthem Jutting, 1962, a microsnail (Fig. 9D), within a portion of the moist rock walls, as well as in rock crevices, but without being sympatric with other millipedes in the same microhabitats.
The habitat preferred by the new species clearly appears to be limestone, especially near caves, although all specimens were found outside the caves, near the entrance zones. No material was collected at twilight, transition or deep zones inside the cave [for a characterisation of the zonal environment in caves, see Liu and Wynne (2019)]. Many of the small holes/caves at the type locality where E. cambodiensis occurs are highly humid and have diminished light, owing to the shade from large trees in the area.
A large concern would be the ongoing habitat destruction very close to the type locality, where a cement factory is located on the opposite side of the mountain. Many North-western Vietnam outcrops in the area appear to have been quarried and it seems plausible that the existence of the type locality would be threatened in the near future. Observation of mating behaviour. Interestingly, all specimens of the new species collected around moist organic material and plants near the caves were pairs of several mating couples (Figs 1C, 9E). No single males or females were found separately. One presumption would be that individual millipedes were perhaps hidden in rock crevices during the daytime. The pairs of the new species mated during the rainy season when the rate of annual rainfall amount is quite high, which may imply the peak in mating occurring around September. The initial observations of the courtship were made by separating seven pairs into individual airflow plastic vials without human disturbance and we found that males appeared to initiate copulation by approaching the female from behind and then slightly reaching to the head region. The male took at least five hours grasping onto a female by its legs before it entwined and finally inserted its gonopod shaft into the female's vulva. Table 3 summarises some significant characters across Eutrichodesmus species.

Notes on species groups in Eutrichodesmus
The genus Eutrichodesmus was recently revised by Golovatch et al. (2009aGolovatch et al. ( , 2009b, who also refined the family and its generic classification, where many remarkable species were also described. Later, Golovatch et al. (2010) reported some sharable characters that can be used for more clearly delimiting species groups. The first and until now only species group, named the "peculiaris-group", was proposed by  and it currently encompasses seven species, viz; E. anisodentus, E. nodulosus, E. pectinatidentis, E. peculiaris, E. silvaticus, E. soesilae and E. taiwanensis, all sharing two rows of tubercles on the metaterga, having a broad and flattened epiproct, lacking a distofemoral process and with complete body volvation. Not only do these morphological traits strongly support this group, but their distribution is also likely to be coherent since most of the species inhabit the same region (southern part of Japan, Taiwan and mainland China).
The discovery of a new species from Cambodia not only represents the first record of the genus, but also of the entire family Haplodesmidae from that country. In this study, we do not only describe a new species, but we also update and compare the morphological characters of all currently known congeners, based on our scrutiny of all relevant original literature sources (Figs 5-8). The comparison, which relies mainly on details of gonopodal structure, body volvation patterns, the number and arrangement of the rows of tubercles and mid-dorsal projections on the metaterga, revealed an adequate delimitation for all 48 remaining species into another group for some coherent assemblages. We assemble 46 species into a second species group, here named the "demangei-group" and the remaining two species which are left ungrouped (see Table 3). Notably, these 46 species share some possibly related characters: metaterga usually with three rows of tubercles (except E. armatus and E. digitatus which have four or more rows); gonopod telopodite            with a distofemoral process (absent from E. astriproximus). All constituent species of Eutrichodesmus are presented in Table 3. The gonopod might be a reliable tool for natural species group delimitations and quite often the assignment of many haplodesmid groups has been based on these characters. Our discrimination has also found the gonopodal structure to be useful in providing several satisfactory characters for sorting out amongst Eutrichodesmus species. Figures 7, 8 clearly show that all species of the "demangei-group" show the same pattern of such gonopodal characters as the existence of a distofemoral process on the telopodite, combined with most species showing three rows of tubercles on the metaterga, while the mid-dorsal projection and body volvation seem to be variable across Eutrichodesmus (Figs 5, 6). In accordance, the distribution of the "demangei-group", which all inhabit Japan, China and mainland Southeast Asia, corresponds to the morphological characters, although their distribution area is obviously larger. The other congeners, E. nodulosus and E. reclinatus, both lack gonopodal information yet, because they were originally described from females only. In spite of their gonopodal structure being unknown, their other morphological traits seem to fit in and serve to place these species in the "demangei-group" much more than to any other group. Whereas the remaining 46 species agree in most respects with the definition of the "demangei-group" given above, there is a strong difference in the structural details of the gonopod, the presence of mid-dorsal projections and the number of the rows of tubercles on metaterga observed in two species, E. communicans and E. reductus. These can be assigned to neither the "peculiaris-group" nor the "demangei-group" due to the remarkable numerous setae without tubercles on the metaterga and the broad distofemoral process on the gonopod femorite, as well as their geographical distribution (E. communicans from Vanuatu and E. reductus from Indonesia) which quite clearly makes them separated from all other congeneric species. Thus, we leave E. communicans and E. reductus amongst ungrouped species as circumscribed above, since they fail to match the definition of the new or other previously-described species groups (see also Table 3).

Discussion
Prior to this study, the millipede fauna of Cambodia consisted of only 23 species, over half of which were described, based on a few specimens from just a handful of locations (Attems 1938(Attems , 1953Likhitrakarn et al. 2015Likhitrakarn et al. , 2020Golovatch 2018). Amongst these, only the polydesmidan families Paradoxosomatidae and Cryptodesmidae have been known to occur in that country (Likhitrakarn et al. 2015;Golovatch 2018).
No micropolydesmoid representative of the family Haplodesmidae has hitherto been reported from Cambodia. This situation is partly remedied herewith by the discovery and description of E. cambodiensis sp. nov. Eutrichodesmus cambodiensis sp. nov. was exclusively found in isolated limestone habitats at or around caves. Based on its apparently highly restricted distribution, the new species can soundly be considered as endemic not only to Cambodia, but also to the Kampong Trach karst. As it is evident from Table 3, almost all Eutrichodesmus species have been found and collected from just one or a few locations confined to small areas. This strongly suggests that they are likely to be endemic to the respective areas and that further micropolydesmoids are most likely to be found in Cambodia.
The new species seems to have partial associations with caves, but it does not tend to show a troglomorphy syndrome because it is pigmented, has no hypertrophied appendages and no specimens have been found living inside the deep cave. Accordingly, this is no troglobite. Nevertheless, certain troglomorphic traits have been suggested in several species of Eutrichodesmus. For example, of the 55 currently known species, 24 are endemic to China alone and over half of these as troglobites, which is definitely a strong concentration of species in the region (Liu and Wynne 2019; Golovatch and Liu 2020). The same tendency to troglomorphy is also marked in most species known from other countries (Hoffman 1977a(Hoffman , 1977b(Hoffman , 1982aGolovatch et al. 2009aLiu et al. 2017).
The mostly thorough work by previous authors has provided sufficiently detailed information on important taxonomic characters that have allowed for species comparisons across Eutrichodesmus to be conducted (Golovatch et al. 2009a(Golovatch et al. , 2009bLiu et al. 2017). Two species groups of Eutrichodesmus are recognisable to account for the wide variety of morphological traits. The "peculiaris-group" was established by Golovatch et al. (2010) and currently accommodates seven species, while 46 species are harboured together in the second, "demangei-group" proposed in this study. Remarkably, the details of gonopodal conformation and the number and arrangement of the rows of tubercles on metaterga support the species assignments to either group. However, although these traits tend to be reliable, two species (E. communicans and E. reductus) could not satisfactorily be assigned into either group and thus remain ungrouped (see Table 3). The new species, E. cambodiensis sp. nov., shows all of its unique characters that are in agreement with its placement in the demangei-group.
In addition to gonopodal morphology, many families of the order Polydesmida prove the great utility of certain surface structures and some other peripheral characters for family-or genus-level classifications (Simonsen 1990;Shear 2008;Mesibov 2009;Akkari and Enghoff 2011). Within Eutrichodesmus, the basic knowledge of periperhal characters for a few old species is very scarce, with no available SEM images. Hence, this requires special attention in the future. With its 55 described species widely distributed in many countries, Eutrichodesmus seems to be the largest group of the micropolydesmoid family Haplodesmidae, but their phylogenetic relationships still remain unknown. Very little can be said about the presumed relationship between the Haplodesmidae and its recent synonym Doratodesmidae, as this synonymy is based solely on a few morphological characters (Hoffman 1982a(Hoffman , 1982bSimonsen 1990;Golovatch 2009a). The further cladistic analysis and a molecular study are the obvious choices to improve the taxonomy by shedding further light on the group's diversity in these millipedes.
The finding of a new Eutrichodesmus species in Cambodia fills in the gap in the distribution of the group across the eastern part of mainland Southeast Asia. As demonstrated recently by the discoveries of micropolydesmoids and other millipedes in the adjacent areas (Likhitrakarn et al. 2010(Likhitrakarn et al. , 2011(Likhitrakarn et al. , 2019, Liu et al. 2014, 2017Golovatch 2018;Srisonchai et al. 2018) with respect to the unexplored and isolated limestone in Cambodia, Malaysia, Myanmar, Laos and Thailand, no doubt further new species remain to be discovered. It is hoped that this work will be a useful contribution to the ongoing process of documenting the diversity of Diplopoda in Cambodia and promote further studies on these remarkable creatures.